1. Field of the Invention
The present invention relates to the identification and microbial susceptibility testing of an unknown microorganism or microorganisms. More specifically, the present invention relates to methods, apparatus, and media for use in the concurrent identification and susceptibility testing of a sample of a microorganism.
2. Description of Related Art
Throughout history, humanity has fallen victim to pandemics of cholera, plague, influenza, typhoid, tuberculosis and other infectious maladies so widespread that few people survived into what is now considered “middle age.” As recently as the 19th century, the average life span in Europe and North America was about 50 years. It was a world in which the likelihood of dying prematurely from infectious diseases was as high as 40%, and where women routinely succumbed to infections during childbirth which are now easily curable by today's standards. In underdeveloped nations, the situation was even worse. Unfortunately, however, unlike many industrialized nations, medical conditions in many underdeveloped nations have never really improved. Indeed, in poorer nations today, infectious diseases, both major and seemingly minor, still contribute to premature death and to the ongoing misery of underprivileged populations.
The emergence of multi-resistant, or “antibiotic-resistant” bacteria, has threatened the security of developed nations and further shaken the citizens of less-developed countries, and is now a worldwide concern. In many nations, antibiotics are used indiscriminately, further contributing to the rise of antibiotic resistance in a variety of bacteria, including species of Enterococcus, Staphylococcus, Pseudomonas, and the Enterobacteriaceae family. The emergence of antibiotic-resistant organisms is very often a result of the over-use of broad-spectrum antibiotics. There is also concern that inappropriate veterinary use of antibiotics may lead to development of antibiotic resistant bacteria. In some cases, these bacteria could then, in turn, infect humans.
The diagnosis of infectious diseases has traditionally relied upon various microbiological culture methods to identify the organism responsible for an infection and then to determine the appropriate antimicrobial treatment for the patient. These methods continue to be important for analysis, despite recent advances in molecular and immunological diagnostics. While the development of rapid and automated methods has served to increase the efficiency of microbiological analysis, traditional quantitative culture methods remain critical for definitive diagnosis of infections. See, Baron & Finegold, Diagnostic Microbiology, 8th ed. C. V. Mosby, (1990), p. 253. Further, these traditional methods are even more valuable in countries unable to afford newer methods, such as automated identification and susceptibility-testing methods. In addition, many areas of the world are devoid of adequate clinical microbiology facilities capable of providing access to newer diagnostic methods. Indeed, in some cases, even traditional culture-based methods are only narrowly available.
Traditional culture-based diagnostic methods share a general set of method steps. A first group of these steps involves the collection and transport of a specimen. The specimen must be material from the actual infection site. Once collected, it is necessary to maintain the sample as near to its original state as possible with minimum deterioration. Transport systems often consist of a protective container, transport medium and a culture swab, as illustrated in FIG. 7. A problem with the use of a holding or transport medium is that it may jeopardize the recovery of certain strains. A major task is to reduce the time delay between collection of specimens and inoculation onto microbiological culture media. The transport container is constructed to minimize hazards to specimen handlers. It is best to minimize adverse environmental conditions, such as rapid changes in pressure, exposure to extremes of heat and cold or excessive drying. The transport of fluid specimens to the laboratory must be done as quickly as possible. It is recommended that a 2-hour maximum time limit be imposed between collection and delivery of specimens to the laboratory. This limit poses a problem for specimens collected any distance from a clinical microbiology laboratory.
In addition to the above difficulties, under some conditions, traditional microbiological culture media suffer from several weaknesses. First, satisfactory microbiological culture media must generally contain many components to successfully support bacterial life. More specifically, satisfactory media must include available sources of water, vitamins, inorganic phosphate and sulfur, trace metals, carbon and nitrogen. These needs may be supplied from a number of sources. In addition, various media may include agents which selectively allow the growth of specific organisms while preventing the growth of others. Media may often include compounds that enhance the ability of a user to identify the bacteria growing thereon. The following is a list of common media constituents with their sources in parenthesis: (1) Amino-nitrogen (peptone, protein hydrolysate, infusions and extracts), (2) Growth factors (blood, serum, yeast extract or vitamins, NAD), (3) Energy sources (sugar, alcohols, and carbohydrates), (4) Buffer salts (Phosphates, acetates and citrates), (5) Mineral salts and metals (phosphate, sulfate, magnesium, calcium, iron), (6) Selective agents (chemicals, antimicrobials and dyes), (7) Indicator dyes (phenol red, neutral red), and (8) Solidifying agents (agar, gelatin, alginate, silica gel, etc.).
Culture media is commonly available in both liquid and solid forms. Solid media provides for the isolation of microorganisms contained in a mixture of different microorganisms. Liquid media, often referred to as “broth”, can provide a nutritionally rich environment which is more accessible to individual cells than solid media. This allows the microorganisms to grow rapidly but does not isolate them from each other. Brain Heart Infusion Broth is one such rich liquid media supplying many of the compounds that the cell would otherwise have to synthesize. This allows the cell to devote more of its energy to growth, which is another reason for their faster growth in liquid media.
A selection of the appropriate solid culture media for microbiological test(s) is generally made according to the particular specimen type. Several hundred standard culture media are commercially available. Various culture media have been developed to serve specific purposes, including the identification of bacteria and antibiotic susceptibility testing. One medium used in antibiotic susceptibility testing is Mueller Hinton agar. The media used as identification testing media can generally be divided into five groups: enriched media, differential media, selective media, differential-selective media, and single purpose media. Enriched media have special additives to support pathogens having fastidious growth needs. Examples of enriched media include sheep blood agar and brain heart infusion broth. Differential media allows the differentiation of groups of microorganisms based on color changes of an indicator (sensitive to a property such as pH) in the culture medium that take place as a result of biochemical reactions associated with microorganism growth. Separating organisms that ferment the sugar lactose, for example, from those that do not, is one example of the utility of differential media.
Selective media support the growth of certain microorganisms of interest while suppressing the growth of others. Azide blood agar is an example. Gram-positive organisms grow on this media whereas gram-negative organisms do not. Differential-selective media combine the characteristics of both selective media and differential media, thus allowing the selective growth and rapid differentiation of major groups of bacteria. These media are widely used in tests for gram-negative bacilli (rods). MacConkey and Hektoen media are examples. Single-purpose media isolate one specific type of microorganism. Bile esculin azide agar is an example of this media. Enterococcus and group D streptococcus grow and cause the formation of a dark brown or black complex in the agar.
In modern microbiology laboratories, every attempt is made to use well-trained personnel, working under close supervision, in the processing of specimens. Errors or misjudgments made during laboratory processing, such as improper choice of culture media, can negate all the expertise one may apply in later processing steps such as the reading and interpretation of cultures. Expert microbiologists may often be caught short in making definitive diagnoses because of the selection and use of inadequate or incorrect media in culturing a specimen.
The equipment required for the primary inoculation of specimens includes several microbiological agar-based media plates and a nichrome or platinum inoculating wire or loop (see FIGS. 8B-8E). Plates currently used in the field generally have a shelf life of from one to two months. Specimens are “streaked out” on the surface of the plates to spread the microorganisms across the surface of the culture medium. This results in isolated colonies. As illustrated in FIGS. 8A-8E the first step in “streaking” is to touch and roll the tip of the swab 84 containing the specimen 116 on the surface of the medium (FIG. 8A). Then, using an inoculating loop 118 that has been flamed to sterilize it (FIG. 9), streak the primary inoculum 116 by spreading it out in the first quadrant (FIG. 8B). Re-sterilize the loop 118 and cool. Streak the inoculum from the first quadrant into the second quadrant (FIG. 8C). Repeat the process for the other two quadrants (FIG. 8D-8E). Incubate the plate following the placement of a lid for 18 to 24 hours. The preceding method is the standard prior art method for isolating microorganisms where at each new streaking they become further diluted until they finally become isolated from one another.
As the isolated microorganisms grow on the solid medium, they form a mass called a colony. This mass of cells originated from a single cell and now may consist of hundreds of thousands of cells. These colonies have distinct characteristics that are a clue in the process of identifying the microorganism (see FIG. 10). The sub-culturing of the isolated colonies to additional media produces pure cultures. The microscopic examination of a suspension of bacteria from a colony reveals (a) cellular morphology, (b) cellular arrangement, and (c) motility. These features (See FIG. 11) add additional pieces to the ID puzzle. A gram stain of the sample may also assist the analyst in getting closer to a characterization of the organism. The gram stain is not foolproof however, and can be occasionally misleading because the staining is frequently dependent upon the age of the colony.
The testing of certain enzyme systems unique to each species provides further clues to the ID of an unknown organism. Another basis for ID is the culture requirements, which include the atmospheric needs of the organism, as well as nutritional requirements and ability to grow on different kinds of media. A further basis of ID in regards to the biochemical characteristics includes the mode of carbohydrate utilization, catalase reactions of gram-positive bacteria and oxidase reactions of gram-negative bacteria. ID to the species level is based on a set of physiological and biochemical characteristics including the degradation of carbohydrates, amino acids, and a variety of other substrates.
Commercial kits perform a number of various biochemical reactions. The results of these reactions can reveal unique patterns for ID. Some systems are automated and others are manual. A problem with manual systems is the limited scope in terms of the organisms they target for ID. Additionally it is necessary to first isolate the organism of interest from other microorganisms in an 18 to 24 hour isolation step as described above before applying the organism to the manual or automated ID system. For example, the manufacturer bioMerieux Vitek® markets the following manual systems (listing the target organisms): API 20C AUX (yeasts), API 20E (Enterobacteriaceae and non-fermenting gram-negative bacteria), API 30 Strep. (Streptococcus and Enterococcus), API Coryne (Corynebacteria and coryne-like-organisms), API 20 NE (Gram-negative non-Enterobacteriaceae), API Rapid 20E (Enterobacteriaceae), and API Staph (Staphylococcus and micrococcus). Judgment must be made by the microbiologist as to which isolate to test and the proper ID system to use. This is another source of possible error.
Current microbial testing methods call for initial isolation and identification of the organism first and then, if deemed appropriate, i.e. where a pathogen is identified, performing an antimicrobial susceptibility test. In addition, the analyst must decide which microorganism is responsible for the clinical disease in mixed cultures. There are a number of different ways of doing antimicrobial susceptibility testing (AST). Two of them are disk-diffusion and micro dilution.
In recent years, there has been a trend toward the use of commercial broth micro dilution and automated instrument methods instead of the disk-diffusion procedure. However, there may be renewed interest in the disk-diffusion test because of its inherent flexibility in drug selection and low cost. The availability of numerous antimicrobial agents and the diversity in antibiotic formularies in different institutions has made it difficult for manufactures of commercial test systems to provide standard test panels that fit every facility's needs. Thus, the inherent flexibility of drug selection provided by the disk-diffusion test is an undeniable asset of the method. It is also one of the most established and best proven of all AST tests and continues to be updated and refined through frequent National Committee for Clinical Laboratory Standards (NCCLS) publications. Furthermore, clinicians readily understand the qualitative interpretive category results of susceptible, intermediate, and resistant provided by the disk test. It is an ideal method when doing manual diagnostic microbiology
The initial isolation step results in colonies formed from a single microorganism. The analyst then transfers like colonies into growth broth. The broth is incubated at 35° C. for 2 to 8 hours until growth reaches the turbidity at or above that of a McFarland 0.5 standard 94. This turbidity is equivalent to 1.5×108 colony forming units (CFU)/ml. McFarland standards are prepared using different amounts of barium sulfate in water. This salt is insoluble in water and forms a very fine suspension when shook. Within 15 minutes of adjusting turbidity, a cotton swab 85 transfers this inoculum to a Standard Susceptibility Dish 122. The entire surface of the Mueller-Hinton plate is swabbed three times; rotating the plate approximately 60 degrees between streaking to ensure even distribution (FIG. 12A). The plate stands for 3 to 15 minutes before AST disk 124 is applied. Apply to the agar surface with a dispenser or manually with sterile forceps. Apply gentle pressure to ensure complete contact of the disk with the agar. (FIG. 12B showing one disk added). Incubate for 16 to 18 hours at 35° C. in an ambient-air incubator.
FIG. 12C illustrates the basic principle of the disk-diffusion method of AST. As soon as the antibiotic-impregnated AST disk 124 is exposed to the moist agar surface, water is absorbed into the filter paper and the antibiotic 128 diffuses into the surrounding medium. The rate of extraction of the antibiotic out of the disk is greater than its outward diffusion into the medium, so that the concentration immediately adjacent to the disk may exceed that in the disk itself. As the distance from the disk increases, however, there is a logarithmic reduction in the antibiotic concentration. If the plate has been previously inoculated with a bacterial suspension, simultaneous growth of bacteria occurs on the surface of the agar. When a critical cell mass of bacteria is reached, the inhibitory activity of the antibiotic is overcome and microbial growth occurs. The time (critical time) required to reach the critical cell mass (4 to 10 hours for commonly tested bacteria) is characteristic of each species but is influenced by the composition of the medium and temperature of incubation. The depth of the agar will affect the lateral extent of antimicrobial diffusion before the critical time is reached because diffusion occurs in three dimensions.
The points at which the critical cell mass is reached appears as a sharply marginated circle (margin 126), of microorganism growth 125, with the middle of the disk forming the center of the circle if the test has been performed properly (see FIG. 12D). The concentration of diffused antibiotic at this margin 126 of growing and non-growing bacteria 127 is known as the critical concentration. This concentration approximates the minimal inhibitory concentration (MIC) obtained in dilution tests. The Minimal inhibitory concentration (MIC) is the lowest concentration of a chemotherapeutic agent that will prevent growth of the test microorganisms. The disk-diffusion test that has become standard in the United States is based on the work of Bauer, Kirby and coworkers. The zone size observed in a disk-diffusion test has no meaning in and of itself. The interpretative standards provided by the NCCLS show the correlation between zone sizes and MICs of those species tested by disk-diffusion method.
FIG. 13 shows a poorly prepared AST plate with objectionable overlapping of the zones of growth inhibition from adjacent disks. FIG. 14 shows a poorly streaked AST plate with uneven growth. The zone margins are indistinct, compromising accurate measurement.
A distinct disadvantage of the above prior art is the total time that it takes from obtaining the culture through performing ID and AST. At least three days transpire before results are available. Another disadvantage is the expense to process the specimen using prior art. A further disadvantage of the prior art is the number of steps involved in performing the tests, which increases the likelihood of human error.
A further disadvantage is raised by the limited shelf life of the agar-based microbiology media that is currently used in the art. Specifically, most currently-available agar-based media have a shelf life of from about one to about two months at most. One problem which reduces the shelf life of such media is syneresis, a condition in which the liquid component of the agar media separates from the gel component. This dramatically reduces the utility of the media by segregating the moisture and nutrients needed in all portions of the agar in a liquid phase, rendering the agar uneven in its ability to support sample growth. This restricts the ability of facilities to maintain an inventory of suitable media and complicates the manufacture, distribution, and sale of diagnostic kits utilizing agar-based media currently known and used in the art.